Flow cytometers are laboratory instruments that are used for identifying the particles present in a fluid and the properties of the particles. Cytometers use the principle of light scattering and fluorescence to generate data pertaining to the particles present in the fluid. The fluid is in some cases hydro-dynamically focused so that the particles line up single file in a relatively small fluid core stream. Thereafter, a laser beam is passed through this fluid core stream. After the laser beam strikes the particles in the fluid core stream, these particles emit fluorescence and reflect or scatter the incident light. The emission, the scattered light, and the reflected light are directed to detectors by using lenses, mirrors or other optical elements.
Some commercially available cytometers use laser sources which are air-coupled to the fluid core stream, i.e., the laser light is not directed to the fluid core stream using fiber optic cables. These cytometers attach one or more laser sources to a mounting plate, which is perpendicular to the fluid core stream. The lasers may be attached to the mounting plate either directly or with the aid of mounting brackets. The fluid core stream passes through a cuvette where a laser beam, emitted by the laser source, is intercepted by the particles in the fluid core stream. The particles reflect, scatter or emit light when the laser beam strikes them.
Most commercial laser sources produce beams with a Gaussian profile. Consequently, the laser source has to be aligned to the fluid core stream to ensure that the particles in the fluid core stream intercept the beam at the center, where the beam is at its highest intensity. Since a Gaussian beam exerts the maximum intensity at its center, cytometers with a Gaussian beam source need to exert precise control over flow, location and diameter of the fluid core stream to ensure that the beam strikes the particles with the maximum intensity. To solve this problem, an elliptical laser beam is used in most cytometers. In an elliptical beam, the change in the beam intensity is less as one moves off center compared to a circular beam. Although elliptical beams have a smaller decrease in beam intensity across their center, the total intensity of the beam at its center is reduced compared to a circular beam.
In cases where the laser light is air coupled, temperature changes within the device affect the laser alignment, based on the method used to mount the cuvette and materials used in the whole assembly. As a result, the alignment of the laser source with the fluid core stream can get altered due to the differential thermal expansion of the components used to mount the laser source and cuvette. Consequently, erroneous results can be generated by the cytometer.
Some of the existing commercially available cytometers use fiber optic cables to reduce the effect of differential thermal expansion. In these cytometers, fiber optic cables are used to deliver the laser beam from the source to the cuvette. The fiber optic cables may be mounted on mounting plates which are parallel or perpendicular to the core stream. This can reduce the thermally induced misalignment. However, this decreases the laser light delivered to the sample due to greater losses in the fiber optic cables.
In light of the foregoing details, there is a need for a laser-architecture for cytometers that can reduce the effect of differential thermal expansion on the alignment of the air-coupled laser sources to the fluid core stream. The architecture should use air-coupled lasers to eliminate the losses caused by the use of fiber optic cables.